ABSTRACT Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach. H. pylori infection is associated with an increased risk of cancer of the distal stomach and peptic ulcer disease. In previous studies, we have conducted detailed analyses of a secreted H. pylori toxin (VacA). The H. pylori genome contains three genes that are distantly related to vacA, and each encodes a protein >250 kDa in size. Similar to VacA, these VacA paralogs are predicted to be secreted by a type V (autotransporter pathway). In contrast to VacA, which has been studied in great detail, thus far there has been very little study of H. pylori VacA paralogs. Hypotheses: The hypotheses of this proposal are as follows: (i) VacA paralogs have important functions in vivo; (ii) the expression of VacA paralogs is transcriptionally regulated; (iii) VacA paralogs are secreted by an autotransporter pathway; and (iv) VacA paralogs modulate interactions of H. pylori with host cells. Study Objectives: The long-term goals of this work are to understand the molecular mechanisms that allow H. pylori to colonize and persist in the human gastric mucosa, to understand the molecular mechanisms by which H. pylori infection leads to the development of gastric cancer or peptic ulcer disease, and to develop effective strategies for the prevention of gastric cancer and peptic ulcer disease. The specific objectives are (i) to analyze VacA paralogs in animal models of H. pylori-induced gastric disease; (ii) to analyze the transcription of H. pylori vacA paralogs; and (iii) to analyze the secretion and functional activities of VacA paralogs. Methods: To investigate the role of VacA paralogs in vivo, we will construct isogenic H. pylori mutant strains. We will then compare the ability of wild-type and mutant strains to colonize the stomach in animal models of H. pylori infection, and we will determine whether these H. pylori proteins modulate the development of gastric inflammation, gastric injury or gastric cancer. We will analyze the transcription of genes encoding VacA paralogs both in vivo and in vitro, and we will elucidate the mechanisms by which the transcription of these genes is regulated. Finally, we will use a combination of proteomic and immunologic methods to analyze the secretion and proteolytic processing of VacA paralogs, and we will compare the interactions of wild-type and mutant strains with cultured gastric epithelial cells. PUBLIC HEALTH RELEVANCE: Helicobacter pylori infection is common among Veterans, and Veterans are at risk for development of H. pylori-induced diseases such as stomach cancer and peptic ulcers. During travel to various parts of the world, Veterans are at risk for acquiring H. pylori strains that confer a relatively higher risk of gastric cancer compared to strains found in the United States. The Veterans Administration currently spends millions of dollars each year for acid-suppressive drug regimens to prevent H. pylori-induced complications. Investigation of the molecular mechanisms by which H. pylori colonizes the human stomach and causes gastric diseases is likely to lead to the development of new approaches for the prevention and treatment of H. pylori infection, which would have important clinical implications for the care of Veterans.